Inter-Active Site Communication Mediated by the Dimer Interface β-Sheet in the Half-the-Sites Enzyme, Thymidylate Synthase

Abstract

Thymidylate synthase (TS) is a dimeric enzyme conserved in all life forms that exhibits the allosteric feature of half-the-sites activity. Neither the reason for, nor the mechanism of this phenomenon is understood. We used a combined NMR and molecular dynamics approach to study a stable intermediate preceding hydride transfer, which is the rate limiting, and half-the-sites step. In NMR titrations with ligands leading to this intermediate, we measured chemical shifts of the apo enzyme (lig 0), the saturated holo-enzyme (lig 2), and the typically elusive singly bound (lig 1) states. ~40 amides showed quartet patterns providing direct NMR evidence of coupling between the active site and probes greater than 30 Å away in the distal subunit. Quartet peak patterns have symmetrical character, indicating reciprocity in communicating the first and second binding events to the distal protomer. Quartets include key catalytic residues and map to the dimer interface βsheet, which also represents the shortest path between the two active sites. Simulations corroborate the coupling observed in solution in that there is excellent overlap between quartet residues and main-chain atoms having inter-subunit cross-correlated motions. Simulations identify five hotspot residues, three of which lie at the kink in the unique β-bulge abutting the active sites on either end of the sheet. Inter-strand cross-correlated motions become more organized and pronounced as the enzyme progresses from lig 0 to lig 1 , and ultimately lig 2. Coupling in the apparently symmetrical complex has implications for half-the-sites reactivity and potentially resolves the paradox of inequivalent TS active sites despite the vast majority of X-ray structures appearing symmetrical.

Caught in Action: X-ray Structure of Thymidylate Synthase with Noncovalent Intermediate Analog

“…The NMR titration experiments with the FdUMPCH 2 H 4 F diligand showed that allosteric changes accompanying diligand binding that may lead to half-of-the-sites reactivity are mostly transmitted through the β-sheet interface and involve changes to the dynamics of the two active sites. 16 Consistent with the NMR titration experiments, there are subtle differences in the strand containing active site nucleophile Cys146 in the inactive monomer B. In monomer B of both Int-B analog structures, the side chain of Phe149 in this strand is in two alternate conformations, and in the (6R)-Int-B analog, Cys146 and His147 both adopt alternate conformations (Figure S4).…”

“…There are also differences between the two protomers in the dynamics of the catalytic Cys146 and adjacent residues. The NMR titration experiments with the FdUMPCH 2 H 4 F diligand showed that allosteric changes accompanying diligand binding that may lead to half-of-the-sites reactivity are mostly transmitted through the β-sheet interface and involve changes to the dynamics of the two active sites . Consistent with the NMR titration experiments, there are subtle differences in the strand containing active site nucleophile Cys146 in the inactive monomer B.…”

Weakening of interaction networks with aging in tip-link protein induces hearing loss

“…Long-range H-bond networks also control the correlated motions among β-strands in β-sheet rich proteins. Such correlated motions respond to physical perturbations by controlling the extent and rate of elastic deformations in a protein 48,[56][57][58][59][60] . Proteins with stronger correlated motions can thus withstand larger perturbations.…”

5-Formyltetrahydrofolate promotes conformational remodeling in a methylenetetrahydrofolate reductase active site and inhibits its activity

“…The ligand-induced conformational change upon CHO-H 4 folate and its noncompetitive inhibition in the NADH:menadione oxidoreductase assay were unexpected, as the change in dimer interface (dimer mode 1) is less extensive (than dimer mode 2); crystallographic evidence shows that CHO-H 4 folate is found only in the active site. Several other folate-binding enzymes, namely DHFR ( 18 ) and TYMS ( 31 , 32 ), have shown that protein motions, in the form of conformational sampling in the active site (DHFR) or the existence of conformational ensembles (both) are crucial not only to catalysis but represent a route through which allostery can emerge. In the case of TYMS, the existence of a ligand-induced β-kink in the dimer interface directly couples the active sites between monomers and explains its observed half-sites reactivity ( 31 ), with an asymmetric dimer assembly structure elucidated ( 32 ).…”

“…Several other folate-binding enzymes, namely DHFR ( 18 ) and TYMS ( 31 , 32 ), have shown that protein motions, in the form of conformational sampling in the active site (DHFR) or the existence of conformational ensembles (both) are crucial not only to catalysis but represent a route through which allostery can emerge. In the case of TYMS, the existence of a ligand-induced β-kink in the dimer interface directly couples the active sites between monomers and explains its observed half-sites reactivity ( 31 ), with an asymmetric dimer assembly structure elucidated ( 32 ). More studies are necessary to determine how noncompetitive inhibition in tMTHFR occurs and could prove invaluable in delineating the catalytic cycle of MTHFR itself.…”